Project Summary Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. According to WHO, each year HEV infects approximately 20 million people worldwide resulting in >44,000 HEV-related deaths. Approximately 5.5% of HEV-infected individuals also developed various neurological diseases such as Guillain-Barr syndrome and neuralgic amyotrophy but the underlying mechanism is unknown. It has been demonstrated that HEV infects various neuronal cells, that circulating HEV in blood exists as quasi-enveloped exosome particles, that HEV infection induces significantly higher level of pro-inflammatory cytokine TNF-?, and that exosomes can readily cross the blood-brain barrier (BBB) under inflamed conditions. The long-term goal is to delineate the mechanisms of HEV-associated neurological diseases. In specific aim 1, we hypothesize that the quasi-enveloped exosome HEV in blood with high level of peak viremia, but not the non-enveloped HEV in bile and feces, cross the BBB during HEV infection with TNF-?- activated inflammatory responses to infect neuronal cells in central nerve system (CNS) leading to neurological disorders. By using an established in vitro BBB model, we will answer these questions: (1). Can quasi-enveloped exosome HEV, but not non-enveloped HEV, readily cross BBB to infect neuronal cells? (2). Do pro-inflammatory cytokines such as TNF-? that was consistently induced in high level during HEV infection facilitate the entry of exosome HEV across BBB? (3). Do HEV infection of or entry through brain microvascular endothelial cells in BBB affect TJ protein gene expression? By completing aim 1, we expect to demonstrate that the quasi-enveloped exosome HEV, but not non-enveloped HEV, readily crosses BBB especially when it is inflamed with TNF-?, and that gene expressions of TJ proteins such as occludin and claudins are increased in response to HEV infection especially when the BBB is inflamed by TNF-?. In specific aim 2, we hypothesize that only the quasi-enveloped exosome HEV in blood with high level of peak viremia can invade CNS causing neurological diseases . This would explain why only 5.5% of HEV-infected individuals developed neurological diseases since most HEV infections had a low level of transient viremia. By using a unique HEV pig model, we will answer these questions: (1). Is there a difference in clinical course of neurological diseases in pigs infected with quasi- enveloped exosome HEV or non-enveloped HEV? (2). Does a high level of viremia in infected pigs correlate with development of neurological diseases? (3). What are the cell types in CNS tissues in which HEV replicate? (4). Do there exist in HEV quasispecies populations in different compartments (feces, blood, and cerebrospinal fluid)? By completing aim 2, we expect to demonstrate that neurological diseases develop in more pigs infected with exosome HEV than with non-enveloped HEV during early stages of infection, and that neurological diseases correlates with pigs with high level of peak HEV viremia. We also anticipate to identify difference in HEV quasispecies population in different compartments, characterize the clinical course of neurological diseases, and identify cell types of CNS tissues in which HEV replicates. The results will have important implications in understanding the mechanisms of HEV-associated neurological diseases.